Membrane vesicles formed from soluble components of the E. coli respiratory chain are capable of ejecting protons in response to a respiratory pulse. The H+/0 ratio in the reconstituted system is 3 to 9% that of the native membrane system. This ratio could not be increased by using a variety of different methods for reconstitution or supplementation with DCCD, succinate transport proteins or coenzyme Q6+ menadione. It was shown that a major reason for the low measured H+/0 ratios is that the vesicles are leaky to protons and to the substrate being oxidized, succinate. The formation of a properly oriented membrane potential was shown by the dependence of proton ejection in the presence of potassium.